Flow cytometry Tutorial | Flow Cytometry Data Analysis | Flow cytometry Gating
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- Опубликовано: 25 июл 2024
- This video lecture explains
1. Principle of flow cytometry
2. Overview of instrumentation of flow cytometry
3. Hydrodynamic focusing
4. Flow cytometric data analysis
5. Flow cytometry gating
6. Forward scatter vs Side scatter plot
7. Forward scatter height vs forward scatter area plot for doublet exclusion
8. Single parameter histograms to identify cells with a particular marker expression
9. Two parameter density plots for further analysis
10. Sequential gating to identify specific T subsets
11. Backgating
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Perfect video! Was struggling in undergrad, but now I understand, especially data analysis section, keep it up and thank you :)
We are glad that this video was helpful to you. Please subscribe our channel and like ans share the video to support us. Thank you
Thank you very much .I’ve been looking for interpretation for about 4 hours and finally found your video.Can’t thank you enough
You are welcome.
Very clearly explained! Awesome video :D
Thank you very much. Please consider subscribing us.
Omg that FSX vs SSC debris explaination helped a ton too!
We are very glad that this video was helpful to you:-)
Data analysis section straight to the point!
Thank you.
Fascinating, thanks so much!
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Hi! Great video, really helped a lot, thank you!
I just had two questions if you don't mind my asking (or do you prefer through the fb page rather than the youtube comment section):
1. what exactly is an isotope control, that you mentioned at 12:30?
2. I loved your explanation of the plot analysis, but I don't quite understand the lines graphed in figure 3c (minute 18:53), would you mind enlightening me a bit more on how they are drawn and what they mean? Is it a gating mechanism?
thank you so much in advance!
Dear Hilde Gunnink, Thank you very much for your feedback. We would be very happy, if you suubscribe our channel to support us. Answers to your questions:
1. Isotype controls are primary antibodies that lack specificity to the target, but match the class and type of the primary antibody used in the application. Isotype controls are used as negative controls to help differentiate non-specific background signal from specific antibody signal
2. These lines are showing four quadrants to separate the four different subgroup of cell populations.
Hope this helped.
Excellent explanation 😊❤️
Thank you.
Feeling smarter now. Thank you!!
You are most welcome.
Excellent explanation. I like it a lot
Glad you liked it!
Excellent explanation 😊
Thank you.
Excellent explanation sir.
Thank you.
Thank you very much 🙏🏾
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Thank you, helped a lot
You are most welcome:-)
it's useful, thanks so much
You're welcome!
Very informative video, thanks!
Thanks a lot.
great video! Could you explain more about granularity and complexity?
How would you know if the qc data passed or failed?
Thank you so much!!
You are welcome.
Thank you for the video but all these gating process are they hypothesis or its for sure that those cell types are as you mentioned Monocytogenes, granulocytes etc. How do I double confirm these cell population subset?
These are the cells as mentioned in the video. However, if you want to double confirm you can perform staining for the specific marker of these cells and check under microscope 🔬
Thank you!!
You are most welcome
Found this video to be very helpful but I was wondering what cells are represented by the Q4 dots in the far right graph at 15:27
These are double negative cells i.e. CD3 and CD19 double negative cells.
Thank you bro!
You are most welcome.
Sorry, could you explain what is doublets? Thanks
Occasionally we would see two cells stuck together referred to as as a "doublet."
thank you :
You are most welcome
Thanks for the walk through! I'm curious. Is your accent Nepali?
Yeah I was thinking the same !!!!
Many thanks for great explanation but
Can I have a question?
What does the colors of the dots indicate (as in 19:00). Is it the density? Like the number of cells in red area will higher than yellow one, and yellow > green > blue right?
sorry for my poor english
Yes, that is correct
@@BiologyLectures thank you!
@@quangvinhnguyen114 You are most welcome
Superrrrrrr
Thank you so much.
Thanks
You are welcome
Good morning. I'm busy with my research project on flow cytometry. I'd like to know what the cut-off ranges are or what percentage range is bright and dim. I need to do a statistical analysis, but I need a cut-off range for the bright and dim results.
Kind regards
Good morning! In flow cytometry, determining bright and dim populations often depends on the specific markers or fluorochromes used in your experiment, as well as the instrument's sensitivity and settings. There isn't a universal percentage range that defines bright and dim cells because it can vary based on several factors.
However, I can provide you with some general guidance on how to set your cutoff ranges:
Use Positive and Negative Controls: Utilize positive and negative controls in your experiment. Positive controls should be strongly expressing the marker of interest, while negative controls should be cells that do not express the marker. These controls can help you establish appropriate gates for bright and dim populations.
Consider Histograms: Analyze histograms of your samples. Histograms can give you an idea about the distribution of fluorescence intensity. You can set gates based on these histograms to separate bright and dim populations.
Use Fluorescence Minus One (FMO) Controls: FMO controls are helpful for setting gates in multicolor flow cytometry experiments. FMOs contain all fluorochromes except the one you're interested in. By using FMO controls, you can establish gates that differentiate specific fluorescence from background or nonspecific staining.
My research is about the CD123 in leukaemia. I did literature review on the flow cytometry part of it and I'm not getting any solution. The statistician asked me to give him the percentage range for bright and dim results, so that he can do the data analysis for me.
I didn't run the flow cytometry, I only obtained the patient results from the system. Based on your response, I'm guessing I'll have to go to the lab and find someone who runs them to check all that you've suggested?
My exams are starting in November, and I need to complete my research write-up before then. 👌🌸
In a density plot, what the do the red, green, blue color signify?
Thank you for raising this question. In density plot, the red color represents the highest density of events within the cell population. With decreasing density, the color transitions from yellow over green to blue
@@BiologyLectures Thanks for the reply. So for example at 8:55, in the lymphocyte population, the red would be the highest density of a certain pop of lymphocytes (say B cells), then yellow would be the area with the second highest density of cell, say T cells? I'm just trying to understand how to read it.
Awesome. Thank you.Debris is pronounced... de-bree... not de-bris
Thanks for your feedback 😊
15:20-19:15
Sir please provide ppt
Too many ads
Presentation would have been better if you had not used too many "oks" almost at every sentences.
Thanks for your feedback. Will improve it.